Systemic treatment with cigarette smoke extract affects zebrafish visual behaviour, intraocular vasculature morphology and outer segment phagocytosis

**Background:** Cigarette smoking is a well-established risk factor for several ocular diseases, including age-related macular degeneration (AMD), cataracts, and dry eye disease. However, the specific mechanisms by which cigarette smoke constituents affect vision and retinal biology remain incompletely understood. This study aimed to investigate the effects of acute systemic exposure to cigarette smoke extract (CSE) on visual function and retinal biology using zebrafish larvae, a vertebrate model with high structural and functional homology to the human eye.

**Methods:** Wild-type zebrafish larvae at 3 days post-fertilization (dpf), when the visual system is mature, were exposed to CSE (10, 15, or 20 μg/mL) for 4, 24, or 48 hours. Visual function was assessed using optokinetic response (OKR) assays (standard, visual acuity, contrast sensitivity, and colour-specific drums) and visual motor response (VMR) assays. General toxicity was evaluated via touch response, heart rate, and swim bladder inflation. Molecular changes were analysed by RT-PCR for oxidative stress and apoptosis genes (cat, gpx1a, nfe2l2a, casp3a) in enucleated eyes. Ocular morphology was examined using light microscopy, fluorescence microscopy (hyaloid vasculature in Tg(fli1:EGFP) larvae), and transmission electron microscopy (TEM) for RPE phagosome quantification.

**Key Results:** - **Visual behaviour:** After 48 hours, CSE-treated larvae showed a dose-dependent reduction in OKR. Larvae treated with 10, 15, or 20 μg/mL CSE exhibited 13.7, 10.7, and 9.4 saccades per minute, respectively, significantly lower than the 19.7 saccades per minute in 0.05% DMSO controls (p=0.0093, p=0.0004, p<0.0001). Visual acuity was impaired at 0.2 cycles per degree (cpd) for 15 and 20 μg/mL CSE (1.6 and 0.9 saccades/min vs. 6.5 in controls, p<0.0001). Contrast sensitivity was reduced at 20% contrast for all CSE doses (e.g., 7.2 saccades/min for 10 μg/mL vs. 17.9 in controls, p=0.0006). Colour-specific OKR showed significant reductions, with the largest percentage reduction for black-blue drums (99.4% at 20 μg/mL). After 24-hour exposure, OKR was also significantly reduced (15.53 vs. 25.41 saccades/min, p<0.0001), and VMR showed reduced overall activity (52.5% reduction, p<0.0001) and reduced peak ON activity (p=0.0342). - **Oxidative stress:** gpx1a expression was significantly increased after 4 hours (p=0.0001) and 24 hours (p=0.0003) of CSE exposure, while cat, nfe2l2a, and casp3a showed no significant changes. - **Hyaloid vasculature:** After 48 hours, 20 μg/mL CSE significantly increased hyaloid vessel diameter from 4.833 μm (controls) to 5.885 μm (p=0.0333) and reduced the number of primary branches (p=0.002). - **Ocular biometry:** Axial length was significantly increased in 20 μg/mL CSE-treated larvae (216.9 μm vs. 205.1 μm in controls, p<0.0001), and lens thickness increased to 107.9 μm vs. 99.65 μm (p=0.0044). Retinal layer thicknesses were not significantly altered. - **RPE phagosomes:** TEM revealed a significant increase in RPE phagosome density in 20 μg/mL CSE-treated larvae (0.1425 phagosomes/μm RPE) compared to controls (0.093 phagosomes/μm RPE, p=0.0002), representing a ~50% increase.

**Clinical Implications:** This study provides the first evidence that acute CSE exposure selectively impairs visual function in zebrafish, mimicking aspects of smoking-related ocular pathology. The observed increase in gpx1a expression indicates oxidative stress, a key driver of AMD and cataracts. The dilation of intraocular vessels aligns with clinical findings of altered retinal vascular calibre in smokers and AMD patients. The increase in axial length and lens thickness may relate to myopia and cataractogenesis, respectively. The elevated RPE phagosome density suggests dysregulation of outer segment phagocytosis, a process critical for photoreceptor health and implicated in AMD. These findings establish zebrafish as a valuable in vivo model for studying cigarette smoke-induced ocular toxicity and for screening potential therapeutic interventions.